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PDGFRalpha/beta and VEGFR2 polymorphisms in colorectal cancer: incidenceand implications in clinical outcome

BMC Cancer 2012, 12:514 doi:10.1186/1471-2407-12-514

Purificacion Estevez-Garcia ([email protected])Angel Castao ([email protected])Ana C Martin ([email protected])

Fernando Lopez-Rios ([email protected])Joaquin Iglesias ([email protected])

Sandra Muoz-Galvn ([email protected])Iker Lopez-Calderero ([email protected])

Sonia Molina-Pinelo ([email protected])Maria D Pastor ([email protected])

Amancio Carnero ([email protected])Luis Paz-Ares ([email protected])

Rocio Garcia-Carbonero ([email protected])

ISSN 1471-2407

Article type Research article

Submission date 22 June 2012

Acceptance date 28 October 2012

Publication date 12 November 2012

Article URL http://www.biomedcentral.com/1471-2407/12/514

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PDGFR/ and VEGFR2 polymorphisms in

colorectal cancer: incidence and implications inclinical outcome

Purificacion Estevez-Garcia1,2

Email: [email protected]

Angel Castao3


Ana C Martin4


Fernando Lopez-Rios5


Joaquin Iglesias4


Sandra Muoz-Galvn1


Iker Lopez-Calderero1,2


Sonia Molina-Pinelo1


Maria D Pastor1


Amancio Carnero1


Luis Paz-Ares1,2


Rocio Garcia-Carbonero1,2,*


1Instituto de Biomedicina de Sevilla (IBIS) (HUVR, CSIC, Universidad de

Sevilla), Sevilla, Spain

2Medical Oncology Department, Hospital Universitario Virgen del Rocio,

Sevilla, Spain

3 Pathology Department, Hospital de Fuenlabrada, Madrid, Spain


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4Bionostra Aplicaciones Biotecnolgicas, S.L.U., Madrid, Spain

5Pathology Department, Centro Integral Oncolgico Clara Campal, Madrid,

Spain

*

Corresponding author. Medical Oncology Department, Hospital UniversitarioVirgen del Rocio, Sevilla, Spain

Abstract

Background

Angiogenesis plays an essential role in tumor growth and metastasis, and is a major target in

cancer therapy. VEGFR and PDGFR are key players involved in this process. The purpose of

this study was to assess the incidence of genetic variants in these receptors and its potential

clinical implications in colorectal cancer (CRC).

Methods

VEGFR2, PDGFR and PDGFR mutations were evaluated by sequencing their tyrosine

kinase domains in 8 CRC cell lines and in 92 samples of patients with CRC. Correlations

with clinicopathological features and survival were analyzed.

Results

Four SNPs were identified, three in PDGFR [exon 12 (A12): c.1701A>G; exon 13 (A13):c.1809G>A; and exon 17 (A17): c.2439+58C>A] and one in PDGFR [exon 19 (B19):

c.2601A>G]. SNP B19, identified in 58% of tumor samples and in 4 cell lines (LS174T,

LS180, SW48, COLO205), was associated with higher PDGFR and pPDGFR protein levels.

Consistent with this observation, 5-year survival was greater for patients with PDGFR B19

wild type tumors (AA) than for those harboring the G-allele genotype (GA or GG) (51% vs

17%; p=0.073). Multivariate analysis confirmed SNP B19 (p=0.029) was a significant

prognostic factor for survival, independent of age (p=0.060) or TNM stage (pG SNP is commonly encountered in CRC patients and isassociated with increased pathway activation and poorer survival. Implications regarding its

potential influence in response to PDGFR-targeted agents remain to be elucidated.

Keywords

VEGFR, PDGFR, SNP, Colorectal cancer, Angiogenesis, Prognosis


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Background

Colorectal cancer (CRC) is the third most common tumour in the world, with over 1.2 million

new cases diagnosed every year, and is responsible for about 8% of cancer related deaths [1].

Approximately one third of patients present metastatic disease at diagnosis, and about 40% of

those with early-stage tumors will eventually relapse at some point over the course of the

disease [2,3]. Although prognosis has greatly improved over the past decades due to

significant surgical and medical advances, once the tumor has progressed beyond surgical

resectability, the disease is essentially incurable and median survival ranges from 14 to 24

months with best available systemic therapy [4]. Development of new more effective agents

is thus actively pursued.

Angiogenesis has become a major target in colorectal cancer therapy. Bevacizumab, a

humanized monoclonal antibody against the vascular endothelial growth factor A (VEGF-A),

was the first antiangiogenic agent to demonstrate efficacy in CRC. In the pivotal study by

Hurwitz et al., the addition of this agent to irinotecan-based combination cytotoxic therapysignificantly improved survival compared to irinotecan-based chemotherapy alone in patients

with advanced CRC [5]. Subsequently, bevacizumab has been tested in combination with

other chemotherapy regimens with more modest results [3,4]. More recently, a benefit in

survival has been also reported in patients with advanced CRC with two new promising

antiangiogenic drugs: aflibercept (a VEGF trap) in combination with FOLFIRI (folinic acid,

5-fluoruracil and irinotecan) following progression to oxaliplatin-based therapy [6], and

regorafenib (a novel tyrosine kinase inhibitor targeting VEGFR, PDGFR, FGFR, RET, KIT

and TIE2) as single-agent therapy in patients who had progressed to all standard therapies

[7]. These results clearly illustrate angiogenesis inhibition is to play a major role in the

management of this disease.

Angiogenesis is a highly controlled process under physiological conditions, such as

embryonal development, postnatal growth and wound healing, but is also a critical driver of

tumor growth and progression [8]. It is tightly regulated by a complex equilibrium among

different pro- and antiangiogenic factors secreted both by tumor cells and by cells of the

tumor microenvironment (pericytes, endothelial, mesenchymal or immune cells). VEGF and

their receptors represent one of the best validated pathways involved in angiogenesis [9,10].

VEGF stimulates both proliferation and migration of endothelial cells, enhances

microvascular permeability, and is essential for revascularization during tumor formation. It

is commonly over-expressed in human tumors, and this is often associated with increased

vascular density and more aggressive clinical behavior. VEGF-A and its main receptor,

VEGFR2/KDR, are key members of this family and common targets of antiangiogenic agents[11,12].

Platelet-derived growth factor (PDGF) and their receptors (PDGFR-, PDGFR- and

PDGFR-) play also a critical role in angiogenesis regulation by exerting important control

functions in mesenchymal cells during development [13]. PDGF is expressed by endothelial

cells and acts in a paracrine manner by recruiting PDGFR-expressing cells, such as pericytes

and smooth muscle cells, to the developing vessels, thus improving pericyte coverage and

vessel function. PDGF signaling promotes cell migration, survival and proliferation and

indirectly regulates angiogenesis by inducing VEGF transcription and secretion [10,13,14].

Mutations involving up-regulation of PDGF and/or PDGFR, as well as PDGFR-dependent

growth stimulation, have been documented in a number of solid tumors and hematological

malignancies, suggesting a likely role of this pathway in carcinogenesis [10,15]. Moreover,


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agents antagonizing PDGFR-mediated signaling have also demonstrated antineoplastic

activity in preclinical models and in clinical trials, including some conducted in patients with

CRC (i.e. regorafenib) [7]. Nevertheless, several other drugs also targeting these pathways

(i.e. sunitinib, sorafenib) [16,17] have failed to prove a significant positive impact on the

outcome of patients with CRC. The biological grounds for these discordant results are not

well understood.

Therefore, and in spite of their undeniable success, only a small proportion of patients do

actually benefit from antiangiogenic agents, and reliable tools to prospectively identify which

patients are more likely to benefit are scarce. In this scenario, efforts to unravel the intricate

molecular pathways governing tumor angiogenesis are certainly needed for progress to be

made. In the present study, we sought to evaluate the incidence of genetic polymorphisms of

some of the key players of angiogenesis, such as VEGFR-2, PDGFR-A and PDGFR-B, and

their potential influence in CRC biology. With this purpose we sequenced the tyrosine kinase

domains of these receptors in 8 CRC cell lines (T84, LOVO, LS174T, HT29, LS180, SW48,

SW480, COLO205) and in 92 tumor samples of patients with colorectal adenocarcinoma.

Correlations of encountered genetic variables with protein expression in cell lines, as well aswith clinicopathological features and survival of these patients were also analyzed to assess

their potential biological and clinical implications.

Methods

Laboratory Procedures

CRC cell lines

Eight human CRC cell lines (T84, LOVO, LS174T, HT 29, LS180, SW48, SW480 andCOLO205) were selected and purchased from the European Collection of Cell Cultures

(ECACC). They were representative of patients with different gender, age and tumor stage.

Cell culture

Each cell line was grown in conditions of temperature, humidity, O2 and CO2 levels, culture

medium and supplements according to providers instructions. Once they reached confluence

in monolayer DNA extraction was performed. The total DNA yield was determined using a

Nanodrop ND-1000 spectrophotometer (Nanodrop Tech, DE, USA).

DNA isolation from human tumor samples and culture cells

Formalin-fixed paraffin-embedded tissues from the 92 selected CRC patients were provided

by the Pathology Departments of the corresponding institutions. Samples were mainly

obtained from the primary tumor (96%), either by surgical (87%) or endoscopic procedures.

Three tissue sections of each tumor were first deparaffinized and rehydrated by serial passes

in D-Limoneno (Histo-Clear, National Diagnostic, Atlanta, GA, USA) and ethanol (100%).

Then, DNA isolation from both human tumor tissue samples and culture cells was performed

with the REAL pure genomic DNA extraction kit (Durviz, Valencia, Spain) according to the

manufacturers instructions and then then purified using ion exchange columns (QIAGEN

Miniprep kit Cat. No. 27106). The total DNA yield was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Tech, DE, USA).


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Genotyping

Public databases including National Center for Biotechnology Information (NCBI)

(http://www.ncbi.nlm.nih.gov), University of California Santa Cruz (UCSC) Genome

Bioinformatics (http://genome.ucsc.edu) and Ensembl Genome Browser

(http://www.ensembl.org/index.html) were reviewed to obtain the haplotypes of the threegenes of interest and their reported genetic variants. The exomic regions corresponding to the

tyrosine kinase domains, which were the regions with the highest probability of mutations,

were then identified for each gene: exons 17 to 26 for VEGFR2, and exons 12 to 21 for

PDGFR and PDGFR. Specific primers were designed to amplify these exons using expert

software in order to minimize non-specific or erroneous amplifications and improve

outcomes. Primers used in this study are described in Additional file 1: Table S1.

Amplification of the tyrosine kinase domains in both CRC cell lines and tissue samples was

performed by a polymerase chain reaction (PCR) method. Fifty nanograms of the genomic

purified DNA were amplified in a PCR reaction containing 1.5 units of DNA polymerase

EuroTAQ (Genycell Biotech Spain SL; Santa Fe, Granada, Spain), 1xEuroTaq buffer, 2.5

mM Mg2+, 0.4 M forward and reverse primers, 80 M dNTPs (20 M each one), 1% DMSOand 1M betaine in a volume of 50 l. The PCR cycling conditions were as follows: initial

denaturation at 94C for 5 minutes, 5 cycles at 94C for 1 minute, and annealing that began at

67C for 45 seconds; this temperature was decreased 2C each cycle to 59C (67, 65, 63, 61,

59) and then 45 seconds at 72C. This was followed by 35 cycles at 95C 1 minute, 55C for

45 seconds and 72C for 45 seconds. The last step was a final extension cycle at 72C for 10

minutes.

DNA sequencing

PCR products were first purified using the microClean kit (Microzone Ltd.; Haywards Heath,UK) or ExoSAP-IT for PCR Product Clean-Up USB (Affimetrix Inc; Santa Clara, CA,

USA) for individual reactions or PERFORMADTV V396-Well Short Plates (Genycell

Biotech Spain SL; Santa Fe, Granada, Spain) for 96 plate reactions. Direct bidirectional

sequencing of the PCR products was done using BigDyeTerminator Cycle v3.1 Sequencing

Kit (Applied Biosystems; Carlsbad, CA, USA) and ABI 3110 Genetic Analyser (Applied

Biosystems) according to the manufacturers instructions. All fragments were double-strand

sequenced a number of times, and genetic variations found were checked twice. Sequencing

analysis was performed using Chromas Lite, Clustal W and DiAlign software.

Analysis of protein expression

Cells were washed twice in 1x PBS, pelleted for 30 seconds at 14000x g and lysed in lysis

buffer (TrisHCl pH 7.5 50 mM, NP40 1%, glycerol 10%, NaCl 150mM, complete protease

inhibitor cocktail, 2 mM; Roche). After centrifugation, supernatant protein extracts were

aliquoted and stored at 80C until use. The amount of protein was determined by Bradford

assay using BSA (bovine serum albumin) as a standard. The appropriate protein quantity was

dissolved in Laemli buffer (TrisHCl pH 6.8 62.5mM, glycerol 10%, SDS 1%, 2-mercapto

ethanol 5%, bromphenol blue 0.0025%) and the proteins were separated in SDS-PAGE gels

(12%) before they were blotted onto Nitrocellulose Transfer membrane (Whatman -

Protrans). Primary antibodies employed were: p-PDGFR- (Tyr1021)-R 1:400 (Santa

Cruz#sc-12909-R), PDGFR- 1:500 (Santa Cruz#sc-339), tubulin 1:10000 (SigmaT6557).

The secondary antibodies used were goat anti-rabbit Alexa Fluor 680 1:5000 (Invitrogen A21057) and donkey anti-mouse IRDye 800CW 1:5000 (Rockland Inc.605-731-002).


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CRC study population, tumor samples and data collection

Patients that met the following inclusion criteria were selected for the present study: (1)

histologically confirmed diagnosis of primary CRC; (2) adequate clinical data recorded in

medical charts; (3) adequate tissue specimen available for additional molecular assays (a

proportion of tumor cells > 50% was required). Cases were reviewed according to apreviously designed protocol which included the following clinical data: age, sex, date of

diagnosis, baseline carcinoembryonic antigen (CEA) plasma levels, primary tumor location,

TNM stage [18], histological type, tumor differentiation, surgical treatment (type and

outcome of surgery), chemotherapy (adjuvant or for advanced disease, regimen used),

radiotherapy (neoadjuvant, adjuvant or palliative), date of last visit or death and cause of

death. The study protocol was approved by the institutional review boards of participating

centers.

Main characteristics of the 92 included patients are summarized in Table 1 and are

representative of a standard CRC population. The median age was 68 years, 63% were male

and 40% presented advanced disease at diagnosis. The great majority had conventional

adenocarcinomas (86%) and only 13% were poorly differentiated tumors. Cancer specific

therapy is outlined in Additional file 1: Table S2. Patients with early stage disease (I-III)

underwent primary tumor surgery with curative intent. Adjuvant fluoropyrimidine-based

chemotherapy with or without oxaliplatin was indicated in patients with high risk stage II or

stage III CRC following surgical resection. Neoadjuvant or adjuvant radiotherapy was added

in stage II-III patients with rectum primaries. Patients with advanced stage IV disease were

managed primarily with systemic chemotherapy that included oxaliplatin- (44%) or

irinotecan-based (13%) combination regimens or fluoropyrimidines alone (3%). With a

median follow-up of 31 months (range: 8 to 99 months), 59 patients (64%) had died due to

disease progression or to complications of cancer therapy.

Table 1Population and tumor samples characteristics

N (%)

Age (years)

Median (range) 68 (4587)

Gender

Female 34 (37.0%)

Male 58 (63.0%)

Primary tumor location

Right colon 27 (29.3%) Transverse colon 5 (5.4%)

Left colon 9 (9.8%)

Sigmoid colon 18 (19.6%)

Recto sigmoid colon 14 (15.2%)

Rectum 19 (20.7%)

Histology

Conventional adenocarcinoma 79 (85.9%)

Mucinous or colloid adenocarcinoma 12 (13.0%)

Signet ring cell adenocarcinoma 1 (1.1%)

Tumor differentiation


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Well differentiated 22 (23.9%)

Moderately differentiated 46 (50.0%)

Poorly differentiated 12 (13.0%)

TNM stage

I 8 (8.7%) II 22 (23.9%)

III 24 (26.1%)

IV 37 (40.2%)

Unknown 1 (1.1%)

Baseline CEA (ng/mL)

High 33 (35.9%)

Normal 59 (64.1%)

Median (range) 3 (013.318)

TNM: Tumor, Node and Metastases; CEA: carcinoembryonic antigen

Statistical analysis

A minimum sample size of 80 patients was planned to be screened in case no mutations were

to be encountered, as in such a case the probability of finding mutations in the general

population was estimated to be very low ( 4.4%; =0.05, =0.80) and therefore non-

clinically relevant. Considering an expected drop-out rate of about 10% (technical issues or

others), 92 patients were finally selected for study entry. Descriptive statistics were used to

characterize the most relevant clinical parameters. The association of categorical clinical or

pathological features and mutation type was explored by the chi-squared test or Fishers exact

test when appropriate. Overall survival (OS) was calculated from the time of histological

diagnosis to the date of death (deaths due to surgical complications were censored). The

Kaplan-Meier product limit method [19] was used to estimate OS, and differences observed

among patient subgroups were assessed by the log rank test [20]. Multivariate analysis using

the Cox proportional hazards model [21] was performed to assess the association between

mutations and clinical outcome while adjusting for other potential confounding factors such

as age, tumor stage, primary tumor location, CEA levels and tumor differentiation. P


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SNPs identified in CRC cell lines

Both SNP A12 and SNP A17 were found in homozygosis in all CRC cell lines. PDGFR-A13

SNP was present in heterozygosis in two cell lines (LS174T and LS180), and PDGFR-B19

presented a SNP in heterozygosis in four of them (LS174T, LS180, SW48 and SW480).

SNPs identified in CRC patient tumor samples

PDGFR-A12 and PDGFR-A17 analysis was feasible in all tumor samples, and all of them

showed the SNPs variants in homozygosis. PDGFR-A13 was successfully analyzed in 73

cases (79%), being the SNP A13 detected in heterozygosis in 18% of analyzed samples (13

patients). PDGFR-B19 complete analysis was achieved in 78 patients (85%), and the SNP

B19 was found in 58% of evaluable samples (45 patients), both in homo- and heterozygosis

(7 and 38 patients, respectively). Figure 1 illustrates DNA sequencing of PDGFR exon 12

and PDGFR exon 19, showing SNPs identified in our population.

Figure 1Electropherogram of a PDGFR sequence. 1a: Three examples of paraffin-embedded tumour tissue DNA sequence analysis of PDGFR exon wild type (Adenine), and

SNP B19 in heterozygosis and homozygosis (A to G transition), respectively. 1b: DNA

sequence of a paraffin-embedded tumour tissue sample presenting PDGFR exon 13 SNP in

heterozygosis (G to A transition)

Correlation of PDGFR and PDGFR genetic variants and

clinicopathological features

Distribution of SNPs A13 and B19 according to gender, age, baseline CEA levels, primary

tumor location, histological type, TNM stage at diagnosis and tumor differentiation isdescribed in Table 2. The only observed correlations that were of borderline statistical

significance were those found between SNP B19 and primary tumor location, and SNP A13

and tumor differentiation. Indeed, the PDGFR B19 SNP was more commonly encountered

among patients with colon primaries than in those with primary tumors located in the rectum

(63.9% vs 35.3%; p=0.051). On the other hand, PDGFR SNP A13 was never detected in well

differentiated tumors, whereas it was identified in 23% of moderately or poorly differentiated

ones (p=0.053).


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Table 2Correlation of PDGFR A13 and B19 mutational status and clinical-pathologicalfeatures

Clinical Features PDGFR exon 13 PDGFR exon 19

WT SNP p WT SNP p

N (%) N (%) N (%) N (%)

Gender 0.754 0.346

Female 24 (85.7) 4 (14.3) 10 (34.5) 19 (65.5)

Male 36 (80.0) 9 (20.0) 23 (46.9) 26 (53.1)

Age 0.127 0.653

68-years old (median) 34 (89.5) 4 (10.5) 18 (45.0) 22 (55.0)

> 68-years old (median) 26 (74.3) 9 (25.7) 15 (39.5) 23 (60.5)

CEA (carcinoembryonic antigen) 1.000 0.813

Within normal range 39 (83.0) 8 (17.0) 21 (41.2) 30 (58.8)

ULN (5 ng/ml) 21 (80.8) 5 (19.2) 12 (44.4) 15 (55.6)

Primary tumour location 1.000 0.051 Colon 47 (82.5) 10 (17.5) 22 (36.1) 39 (63.9)

Rectum 13 (81.3) 3 (18.8) 11 (64.7) 6 (35.3)

Tumour histology 0.401 0.221

Conventional adenocarcinoma 52 (83.9) 10 (16.1) 30 (45.5) 36 (54.5)

Mucinous or colloid adenocarcinoma 8 (72.7) 3 (27.3) 3 (25.0) 9 (75.0)

TNM stage 0.196 0.170

I-II 23 (92.0) 2 (8.0) 8 (30.8) 18 (69.2)

III-IV 37 (78.7) 10 (21.3) 24 (47.1) 27 (52.9)

Tumour differentiation 0.053 0.586 Well differentiated 15 (100) 0 (0.0) 7 (36.8) 12 (63.3)

Moderately or poorly differentiated 37 (77.1) 11 (22.9) 23 (47.9) 25 (52.1)

Surgery of primary tumor 0.578 0.389

Yes 55 (80.9) 13 (19.1) 32 (43.8) 41 (56.2)

No 5 (100.0) 0 (0.0) 1 (20.0) 4 (80.0)

Surgery of metastasis 0.953 0.451

Yes 13 (86.7) 2 (13.3) 9 (50.0) 9 (50.0)

No 47 (81.0) 11 (19.0) 24 (40.0) 24 (40.0)

Adjuvant chemotherapy 0.295 0.986

Yes 28 (87.5) 4 (12.5) 14 (42.4) 19 (57.6)

No 32 (78.0) 9 (22.0) 19 (42.2) 26 (57.8)

Chemotherapy for advanced disease 0.683 0.929

Yes 36 (83.7) 7 (16.3) 18 (41.9) 25 (58.1)

No 24 (80.0) 6 (20.0) 15 (42.9) 20 (57.1)

PDGFR and PDGFR genetic variants and colon cancer survival

Overall survival of patients according to PDGFR-A13 and -B19 SNPs identified is depicted

in Table 3. No significant impact in overall survival was observed for SNP A13. On the

contrary, 5-year survival of patients PDGFR-B19 WT was substantially greater than thatobserved in those harboring the SNP (51% vs 17%; p=0.073) (Figure 2). Multivariate


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analyses showed the presence of the B19 SNP variant was a significant independent predictor

of survival (HR 2.89, p=0.029). Other variable that retained independent prognostic value in

the Cox regression model was TNM stage (pG,

rs1873778; exon 13 (A13): c.1809G>A, rs10028020; and exon 17 (A17): c.2439+58C>A,

rs2412559] and one in PDGFR [exon 19 (B19): c.2601A>G, rs246395]. SNP B19, present

in 4 CRC cell lines (LS174T, LS180, SW48, COLO205) and in 58% of patients, had a

substantial impact on overall survival, with 5-year survival rates of 51% for patients with

PDGFR B19 wild type tumors versus 17% for those harboring the SNP variant (c.2601A>G).

This is the first study to analyze the PDGFR genotype in a series of human colorectal cancer

and its correlation with different clinicopathological features, and to demonstrate a significantassociation of a PDGFR SNP with patients outcome.


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Angiogenesis is a complex process controlled by a number of interconnected signaling

pathways, among which PDGF and their receptors play a critical role. Moreover, PDGFR has

been the target for many newly developed anticancer drugs, some of them with proven

efficacy in CRC (i.e. regorafenib) [7] and some that have failed to demonstrate a benefit in

patients with this tumor type (i.e. sunitinib, sorafenib) [16,17]. Despite this, however, only

few studies have analyzed the clinical implications of PDGF/PDGFR expression in colorectalcancer. In this regard, Schimanski and cols reported that specific receptor tyrosine kinases

(TK) were overexpressed in K-ras mutated CRC [22]. In particular, VEGFR1, VEGFR2 and

PDGFR expression, documented in 95%, 46% and 62% of tested samples, respectively,

were significantly linked to K-ras codon 12 or 13 mutations. Whether this could translate into

a higher likelihood of responding to TK inhibitors, however, is a matter of speculation. On

the other hand, Wheler et al. reported, in a series of 99 human colorectal carcinomas, that co-

expression of PDGFR/, observed in 57% of tumor samples, was significantly associated

with lymphatic metastasis (P=0.007) and advanced tumor stage (P=0.03) [23]. Similarly, high

PDGFR tumor stromal expression significantly correlated with more aggressive clinical

behavior in patients with breast cancer, including high histopathological grade, estrogen

receptor negativity, high HER2 expression and shorter survival [24].

Nevertheless, PDGFR genetic variants had never been previously assessed in CRC patients.In our study, four genetic variants were identified, all of them corresponding to SNPs

previously reported in public databases. Three of them were silent mutations (A12, A13 and

B19) and the other one was an intronic insertion (A17). PDGFR exon 12 SNP (rs1873778),

present in homozygosis in all CRC cell lines and 100% of analyzed tumor samples, has been

also described in other neoplasias although in a smaller proportion of patients, including KIT

and FLT3 mutation-negative core binding factor (CBFL) acute myeloid leukemias (14% of

35 patients) [25], cervical adenosquamous carcinomas (30% of 30 patients) [26] and gliomas

(7% of 86 patients) [27]. In this last study, no association was found between the presence of

this mutation and PDGFR tissue expression. Our results are in agreement with the

distribution reported for a European Caucasian population at the NCBI website

(http//:www.ncbi.nlm.nih.gov/sites/entrez/), being the G-allele the most frequently

encountered (p=0.98). PDGFR exon 13 SNP (rs10028020), detected in heterozygosis in 2

(LS174T and LS180) of the 8 cell lines examined and in 18% of tumor samples, was

associated with poorer tumor differentiation but no significant correlation was found with

survival. This polymorphism had been first reported also in heterozygosis by Trojani et al. in

34% of CBFL acute leukemias [25], although potential association of this genotype with

clinical features or patients outcome was not explored by these authors. Finally, neither

PDGFR exon 17 SNP (rs2412559), identified in all of our patients, nor PDGFR exon 19

SNP (rs246395), present in 58% of them, had been previously described in human cancers.PDGFR B19 SNP has been reported to be present in the general population with a frequency

of 37%, and was more commonly encountered in our study population among colon primary

tumors (64%) than in tumors of rectal origin (35%). Of note, and despite not being an

activating mutation, the B19 SNP was found to be a significant prognostic factor (HR: 2.89,

p=0.029) independent of tumor stage or patients age. This negative effect on patients

survival did not differ according to primary tumor location (data not shown).

That the identified SNP in exon 19 of PDGFR may indeed have relevant biological

implications is further supported by the fact that analysis of protein content in cell lines

demonstrated the presence of the B19 SNP clearly correlated with higher protein levels of the

PDGF receptor , also in its phosphorylated state. PDGF pathway constitutive activationmaintains highly active MEK, thus phosphorylating Bad and inhibiting apoptosis [14,15].


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Increased PDGF pathway activation has been also shown to contribute to drug resistance by

activating the PI3K pathway [14,15]. Whether or not the presence of this SNP may portend

particular sensitivity to PDGFR-targeted agents is a matter of speculation but certainly

deserves further investigation due to its relevant potential clinical applications.

On the contrary, no relevant findings were identified in our series regarding VEGFR2 TKdomain SNP analysis. As in other solid tumors, overexpression of VEGF mRNA and protein

has been associated with tumor progression and poor prognosis of colon carcinoma [28]. The

VEGF-A gene is known to be highly polymorphic and harbors numerous SNPs, especially in

the promoter, 5- and 3-untranslated regions (UTR), which contain key regulatory elements

that are sensitive to hypoxia [29]. These SNPs contribute to the high variability in VEGF

production among tissues and have been associated with cancer susceptibility, progression,

and anti-VEGF therapeutic response in subjects with a variety of solid tumors including

colorectal cancer. For example, the 936 T-allele has been associated with increased risk of

CRC, advanced stage of disease and worse prognosis, whereas the 634 C allele was

predictive of decreased risk and improved survival. SNPs have also been identified in the

VEGF receptor genes, although the literature in this topic is still very sparse. Very recently,the VEGFR-1 319 C/A SNP, located in the promoter region of the gene, has been reported to

be associated with response to therapy in a cohort of 218 CRC patients treated with differentbevacizumab-containing regimens [30]. In this study by Hansen et al., response rates were

significantly higher in patients homozygous for the A-allele (AA) than in patients with the C-

allele genotype (CC or CA) (56% vs 39%, p=0.015). Similar results were also documented in

bevacizumab-treated pancreatic cancer patients [31]. In addition, functional relevance has

been demonstrated for several SNPs in the VEGFR-1 and VEGFR-2 genes, particularly SNPs

1192C/T (V2971I; rs2305948) and 1719T/A (H472Q; rs1870377). These SNPs are located in

exons 7 and 11, and lead to amino acid changes potentially interfering with the receptors

binding affinity to VEGF-A. In the current study, however, we aimed to explore potential

genetic variations in the TK domain of the VEGFR-2 (exons 17 to 26), which would be

expected to have relevant functional consequences. No mutations were however detected in

our study population in these gene domains.

Identification of relevant SNPs in critical genes involved in angiogenesis may therefore

become valuable tools in assessing risk or predicting cancer response to therapy or prognosis.

However, no consensus exists at present regarding the use of any of these for clinical

decisions as many studies have reported diverging, conflicting or inconclusive results.

Multiple reasons may be responsible for these discrepancies, including gender and interethnic

differences in the distribution of alleles, heterogeneous study populations and small sample

sizes, different sources of DNA (i.e., tumor vs germline) and different methods for SNPanalyses, lack of corrections for multiple testing, links to other loci in the gene or related

genes responsible for the observed effect, bias due to post-transcriptional gene regulation, or

simultaneous presence of somatic or epigenetic changes that may influence outcome.

Prospective validation in appropriately sized and controlled studies is therefore required

before these genetic variants may be used in clinical practice.

Conclusion

In conclusion, the present study has identified, for the first time, PDGFR genetic variants

with relevant clinical and biological implications. In particular, the G-allele genotype of

PDGFR exon 19 SNP (rs246395), which was commonly encountered in our series of CRC

patients (58%), was associated with increased pathway activation and poorer survival.


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Further studies to assess the functional consequences of this genetic variant, as well as to

validate its role as a prognostic marker in this disease are certainly warranted. Implications

regarding its potential influence in response to PDGFR-targeted agents remain to be

elucidated.

Competing interest

The authors declare no conflict of interest.

Authors contributions

Conception and design: RGC, ACM, LPA. Molecular genetic and protein analysis in human

samples and cancer cell lines: ACM, JI, SMP, MDP, SM. Pathological assessment: AC, FLR.

Collection and assembly of data: ACM, JI, ILC, SMP, MDP, SM, PEG, RGC. Data analysis

and interpretation: RGC, LPA, ACM, JI, AC, PEG. Manuscript writing: PEG, SMP, AC,

LPA, RGC. Final approval of manuscript: All authors.

Novelty and impact of present research

This study has identified, for the first time, PDGFR genetic variants with relevant clinical

and biological implications in colorectal cancer. In particular, the G-allele genotype of

PDGFR exon 19 SNP (rs246395), encountered in 58% of tumor samples from colorectal

cancer patients, was associated with increased pathway activation and significantly poorer

survival.

Acknowledgements

We would like to thank the technical staff from Bionostra for technical assistance. This work

was supported by a grant of the Ministerio de Ciencia y Tecnologia of Spain (FIT-010000-

2006-45). PEG is funded by a Rio Hortega grant (09/00207) from the Instituto de Salud

Carlos III (ISCiii), Ministerio de Sanidad, Spain. SMP is funded by the ISCiii (CD1100153),

Fundacin Cientfica de la Asociacion Espaola Contra el Cancer (AECC), Consejeria de

SaludJunta de Andalucia (PI-0224/2009) and Fundacion Mutua Madrilea (2009). MDP is

funded by the ISCiii (CD0900148). LPA is funded by the ISCiii (PI081156 and PI1102688),

Consejera de Innovacion, Ciencia y EmpresaJunta de Andalucia (P08-CVI-04090) and the

75th

Anniversary Roche Spain Fellowship. RGC is funded by the ISCiii (PI 10.02164).

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Additional file

Additional_file_1 as DOCAdditional file 1 Supplementary Tables


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Figure 1


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Time (months)

12096724824

1,0

0,8

0,6

0,4

0,2

0,0

SNP-censoredWT-censoredSNPWT

PDGFR B19

HR=1.93P=0.073

Overall Survival by PDGFR exon 19 Genotype

Figure 2


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Figure 3


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